Chromone-based monoamine oxidase B inhibitor with potential iron-chelating activity for the treatment of Alzheimer’s disease

Abstract Based on the multitarget-directed ligands (MTDLs) strategy, a series of chromone-hydroxypyridinone hybrids were designed, synthesised, and evaluated as potential multimodal anti-AD ligands. Prospective iron-chelating effects and favourable monoamine oxidase B (MAO-B) inhibitory activities were observed for most of the compounds. Pharmacological assays led to the identification of compound 17d, which exhibited favourable iron-chelating potential (pFe3+ = 18.52) and selective hMAO-B inhibitory activity (IC50 = 67.02 ± 4.3 nM, SI = 11). Docking simulation showed that 17d occupied both the substrate and the entrance cavity of MAO-B, and established several key interactions with the pocket residues. Moreover, 17d was determined to cross the blood–brain barrier (BBB), and can significantly ameliorate scopolamine-induced cognitive impairment in AD mice. Despite its undesired pharmacokinetic property, 17d remains a promising multifaceted agent that is worth further investigation.


Introduction
Disease-modifying therapy for Alzheimer's disease (AD) is far from satisfactory 1 . The underlying pathogenesis of AD remained frustratingly under debate despite significant efforts over the last 50 years 2,3 . Interrelated and multifaced etiopathology among AD pathological factors was proposed, especially for iron dyshomeostasis and abnormal MAO-B activities [4][5][6] . Elevated iron has been proved to deposit within the vulnerable neuronal populations and potentiates oxidative stress via the Fenton-and Haber-Weiss reactions, as well as by increasing lipid peroxidative stress [7][8][9] . Increased neuronal iron in AD is known to enhance Ab production and oligomerisation, further facilitating tau dysfunction and neurofibrillary tangles 10,11 . As such, human monoamine oxidases (MAOs) are flavin adenine dinucleotide (FAD)-containing enzymes responsible for the oxidative deamination of monoamine neurotransmitters, and the elevated MAO-B activity led to a higher level of neurotoxic byproducts accelerates neurotransmitters consumption, and neuronal damage 12,13 .
Recently, following the "one molecule, multiple target" paradigm, the emerged multitarget-directed ligands (MTDLs) strategy has been suggested as a powerful and promising alternative paradigm for developing effective anti-AD agents 14,15 . MAO-B has been paid increasing attention as the multifunctional anti-AD agent design target as a consequence of the neuroprotective and neurorescue effects of MAO-B inhibitors 16 . Currently, dozens of MAO-B inhibitors with auxiliary beneficial properties or target affinity (e.g. antioxidative ability, inhibit Ab aggregation, AChE inhibition, and metal chelation) have been developed by integrating pharmacophores or scaffolds from two or more molecules and were proved to undergo unique anti-AD mechanisms ( Figure 1) [17][18][19][20][21] .
Several pieces of evidence reported by our research group have revealed that coumarin/benzamide-based multi-target ligands with the iron-chelating ability and MAO-B inhibitory activity have an attractive anti-AD potential 20,22 . Aiming at identifying new multipotent ligands, in this work, a new series of chromonehydroxypyridinone hybrids were rationally designed by linking chromone core and hydroxypyridinone moiety with the appropriate linkers ( Figure 2). Since studies have demonstrated that 3-carboxyl chromone is a privileged scaffold of MAO-B and possesses specific selectivity over MAO-A isoform, accordingly, the hydroxypyridinone fragment was assembled to 3-position of chromone core [23][24][25] . From the induce-fit perspective, the flexible ethylenediamine linker was chosen to accommodate the hydrophobic cavity as well as partially diminish the rigidity of the molecule 12,26 . Modifications on the 7-position of the chromone ring were also investigated since studies prove that benzyloxy decoration can promote selectivity and efficacy 27,28 . Thus, we synthesised and evaluated chromone-hydroxypyridinone hybrids to explore their bioactivity in association with AD.

Results and discussion
Chemistry According to the difference in linkers, two series of chromone derivatives were outlined: (I) amide-bonded compounds, and (II) C--N bonded compounds. The amide-bonded chromone derivatives (8a-e, 15a-g) were efficiently obtained according to the procedure shown in Schemes 1-3. Intermediate 2 was synthesised starting from commercially available maltol through the protection of the 3-hydroxyl group. Subsequently, the protected maltol was converted into intermediate 3 by reaction with ethylenediamine under reflux conditions (Scheme 1).
Appropriate chromone-carbaldehydes (5a-e) were synthesised by corresponding acetophenone through the Vilsmeier-Haack reaction 23 . Afterward, the chromone carboxylic acids (6a-e) were prepared by Pinnick oxidation, in the presence of sodium chlorite 29 . The chromone-carboxamide compounds (7a-e) were obtained through a reaction that included the generation of an acyl chloride intermediate and the subsequent addition of intermediate 3 (Scheme 2).
The synthetic pathway followed to obtain the first series of compounds (15a-g) involved seven steps (Scheme 3). Except for the difference in acyl chloride reagents, the synthetic procedure of compound 13 was the same as that of the chromone-carboxamide derivatives (7a-e). Subsequently, the ester group of compound 13 was hydrolysed with potassium carbonate and treated with appropriate benzyl bromide to afford the compounds (14a-g).
In turn, the second series of compounds (16a-l) were synthesised by reductive amination, starting from chromone-carbaldehydes (5a-l) to yield the imine intermediate (Scheme 4). Finally, the chromone-based compounds (8a-e, 15a-g, and 16a-l) were performed by a selective debenzylation reaction with boron trichloride to remove the 4-methoxybenzyl protecting groups.

Iron-chelating ability test
Recently, iron has been determined as a pro-oxidant involved in oxidative damage due to Fenton-and Haber-Weiss reactions and ferroptosis 30 . To evaluate the iron chelating capacity of chromone derivatives, all of the compounds were then subjected to determine the pK a values and iron affinity constants for iron (III) by an automated spectrophotometric titration system 20 . The obtained data are provided in Table 1. All derivatives displayed impressive pFe 3þ values ranging from 15.87 to 19.08 (Table 1). Compound 8e revealed the most potent iron-chelating ability (pFe 3þ ¼ 19.08). Overall, no discernible difference in iron-chelating activity exists between the two series of compounds (compounds 8a-e, 15a-g, and 17a-l), both of which are outstanding.
Typically, 3-hydroxypyridin4-one derivatives are bidentate ligands (3:1 complex) and their proton ionisation characteristics, the UV spectrums yielded two pKa values (pK a1 < 3.40, pK a2 ¼ 9.32 À 10.05). The 2D UV titration profile of 17d over the pH range 1.3-11.0 (Figure 3(A)) yielded two pKa Values, 2.86 and 9.78 demonstrating the pH dependence of the ligand ionisation equilibrium (Figure 4(B)). Repeated the titration in the presence of iron (III), over the pH range of 2.1 À 9.0, the visible spectrum reveals three species, FeL, FeL 2 , and FeL 3 , respectively, in the solution system.   The spectral resolution of these three substances led to the determination of the corresponding three equilibrium constants (compound 17d): Log b 1 (13.50), Log b 2 (25.21) ( Figure 5(A)), and Log b 3 (35.13) ( Figure 5(B)), which were the basis for calculating the final pFe 3þ value. The abundance of each species varied with pH as illustrated in Figure 5(C). Table 1, all of the compounds well inherited the iron-chelating ability of the hydroxypyridinone pharmacophore. Compounds with 6,7-alkyl and 6,7-alkoxy substitutions on the benzene ring showed better iron chelation than benzyloxy substitutions. Meanwhile, amide bonds linked compounds exhibited better iron chelation than C-N bond-linked derivatives.

HMAO-B/a inhibition assay and selectivity
Subsequently, the MAOs inhibitory activity of the compounds was measured, as shown in Tables 2 and 3. As detailed below, generally, hybrids with C-N bond linkers were more potent than those amide bond-linked compounds, with most of them having inhibition rates over 40%. Chromones bearing substituents in position C-7 of chromone exhibited better MAO-B inhibitory activity with IC 50 values ranging from micromolar concentration to nanomolar concentration. Furthermore, when the benzyloxy group was inserted into the C-7 of chromone, the inclusion of electron-withdrawing substituents (F, Cl) in the benzyloxy counterposition demonstrated higher activity than interposition replacement. The most promising chromone derivatives, on the other hand, are changed by alkyl groups at C-6 or C-7 of chromone nuclear (compounds 17a-e and IC 50 values range from 67 to 88 nM). Among them, compound 17d showed the best inhibitory activity compared with the reference drug (Pargyline).
Further, the inhibitory activity of compounds against hMAO-A isoform was tested and calculated as a selectivity index [Selectivity index (SI): IC 50 (hMAO-A)/IC 50 (hMAO-B)]. As indicated in Table 3, all compounds demonstrated varying degrees of selectivity, with compound 17b exhibiting high selectivity for MAO-B. Comprehensively considering the iron-chelating ability and the inhibitory activity over enzyme isoforms, compounds 17a and 17d could be performed in subsequent assays.

PAMPA-BBB assay
Good blood-brain barrier (BBB) permeability is the key to exerting drug efficacy in the treatment of central nervous system diseases. To evaluate whether the target compound could penetrate BBB, a parallel artificial membrane permeability assay (PAMPA) was used to predict 32,33 . As outlined in Table 4, The P e value of compound 17a measured by this method was 1.22 ± 0.22 Â 1 0 À6 cm/s (CNS-), indicating that compound 17a had poor permeability and could not penetrate the BBB. However, the P e value of compound 17d was between 2 and 4, suggesting that compound 17d had the possibility of penetrating the BBB. To further confirm these results, a prediction platform (ADMETlab) was selected for verification. Both compounds displayed the desired CNS drug-like properties (MW 450, HBD 5, HBA 10, and Log p 5) and were predicted to pass through the BBB, which is classified as BBBþ. Therefore, due to its potential ability to cross the BBB, compound 17d was selected for subsequent assay.

Kinetics study and cytotoxicity assay of compound 17d
To examine the interaction mode of 17d on MAO-B, kinetic studies were carried out. From the Lineweaver-Burk plots ( Figure 5(B)), we observed that 17d operated by a non-competitive inhibition mechanism. Using GraphPad Prism, the Michaelis constant (K m ), the maximal velocity (V max ) and the inhibition constant (K i ) were calculated (K m ¼ 117.7 lM, V max ¼ 1701, and K i ¼ 74.71 nM). Subsequently, the potential toxic effect of 17d was investigated The data from the reference tested in a 0.1 M KCl solution 31 . c The compounds were tested in DMSO: KCl (0.1 M) ¼ 1:1.5 (v/v) to address the solubility issue.

Molecular modelling
The potential binding conformation of compound 17d in MAO-B (PDB: 2V5Z) was investigated by molecular docking simulation in Discovery studio version 4.0 (BIOVIA, USA). As shown in Figure 6, 17d occupied both the entrance and substrate cavities of MAO-B with the 7-methoxy-chromone ring oriented towards the highly hydrophobic entrance cavity and formed Pi-Pi interaction with TYR 326. Moreover, the chromone-carbonyl oxygen established two hydrogen bonds with residues TYR 326 (3.3 Å) and GLN 206 (3.0 Å). Accordingly, The pyridinone moiety of 17d occupied the substrate cavity in front of the FAD cofactor. Pi-Pi interaction was observed between the pyridinone plane and TYR 435, and a hydrogen bond interaction was monitored between pyridinonecarbonyl oxygen and the residue TYR 188. The above binding mode indicated significant interactions between pyridinone moiety and surrounding amino acid residues, indicating that pyridinone moiety, not only provided chelating ability but also functioned as a critical piece in binding with MAO-B.

Cognitive and memory assays in vivo
Towards this end, compound 17d exhibited favourable multipotent activity. To further explore the in vivo anti-AD effect of compound 17d on the scopolamine-induced cognitive impairment mice model, the Morris Water Maze experiment was selected; data are shown in Figure 7 and Table 5.
Furthermore, the latency to target was significantly shortened when treated with 17d (14 ± 1 vs. 42 ± 6, ÃÃÃ p < 0.001). In terms of the distance to the first entry (Figure 7(C)), mice of the pargyline group led to a remarkably shorter distance to the first entry (2.6 ± 0.3 vs. 8.9 ± 1.2, ÃÃÃÃ p < 0.0001). The group of 17d exhibited a comparable activity to pargyline (3.3 ± 0.3 vs. 2.6 ± 0.3). According to the trajectory diagram (Figure 7(D)), the trajectory of pargyline, memantine, and compound 17d were very clear. In contrast, the trajectory of the model group was very chaotic, which demonstrated that the scopolamine-induced cognitive impairment was significantly ameliorated by compound 17d. As illustrated in Figure 8, except for the control group, all groups of mice showed a significant decrease in body weight after administration on the first day. However, the body weight of mice in group 17d increased after ten days, partially indicating that 17d had no obvious cytotoxicity.

Pharmacokinetic property and BBB permeability investigation
UHPLC-MS/MS method was adopted to investigate the pharmacokinetic profiles of its metabolites after intravenous (i.v.) administration (2.34 mg/kg) and intragastric (i.g.) administration (15 mg/kg) of 17d in the tail vein of rats. The main plasma pharmacokinetic parameters are listed in Table 6. As we can see, compound 17d has higher bioavailability in the case of gavage administration (F ¼ 9.25%), and can be absorbed quickly (T max ¼ 0.25 h) after i.g. administration. Although shorter half-time and lower brain districts, the above results indicated that 17d would be instructive for further study.

Conclusion
Herein, a novel series of chromone-pyridinone hybrids as dual-target-directed agents were designed and synthesised by pharmacophore fusion strategy. The designed derivatives displayed preferable biometal chelating effects and anti-MAO-B activities. Compound 17d was proved to be the most potent MAO-B inhibitor (hMAO-B IC 50 ¼ 67.02 ± 4.3 nM), outperforming the reference drug pargyline (hMAO-B IC 50 ¼ 111.3 ± 0.6 nM). The molecular docking research revealed that 17d may bind to both the substrate cavity and the entrance cavity of MAO-B. Furthermore, compound 17d was demonstrated as a noncytotoxic agent and was able to considerably ameliorate cognitive dysfunction in a scopolamine-induced mice cognition-impaired model. Despite its Table 3. IC 50 and SI for the chromone derivatives on the enzyme activity of MAO isoforms. Comp.

General information
Unless otherwise indicated, all reagents and solvents were used without further purification acquired from commercial suppliers. NMR spectra were acquired on Bruker and Varian spectrometers at 600, 400 MHz for 1 H and 150, 100 MHz for 13 C, respectively. Melting points (m.p., uncorrected) were measured with a B€ uchi B-540 m.p. apparatus. High-resolution mass spectra (HRMS) were   recorded with a Shimadzu LCMS-IT-TOF mass spectrometer equipped with an electrospray ionisation (ESI) source. Routinely, the procedure of reactions was monitored on silica gel by thinlayer chromatography. The purity of the final compounds (>97%) was verified by high-performance liquid chromatography (HPLC) equipped with a UV-diode array detector.

General procedure for the preparation of intermediate 4f-l
Anhydrous potassium carbonate (7.5 mmol) and compound 9 (5 mmol) were dissolved in acetone (20 ml), and appropriately substituted benzyl bromide derivatives (5.5 mmol) were added to the mixture and refluxed for 8 h. Upon completion, the solution of reaction was filtered and the filtrate was removed under reduced pressure. The target compounds were purified by recrystallisation with MeOH/DCM to yield solid powder 4f-l. General procedure for the preparation of intermediates 5a-l and 6a-e A solution of compounds 4a-l (10 mmol) in anhydrous DMF (20 ml) was stirred at À10 C for 35 min. Subsequently, phosphoryl chloride (20 mmol) was added dropwise for 20 min at À10 C. Then the reaction mixture was allowed to stir at room temperature for another 15 h before being poured into ice water. The produced residue was filtered and washed with ethyl ether to obtain solid powders 5a-l. Afterward, a mixture of compounds 5a-e (3 mmol) in methylene chloride (40 ml) and sulphamic acid (15 mmol) in water (36 ml) was cooled to À0 C. Sodium chloride (12 mmol) was dissolved in water (26 ml) and added dropwise to the mixture slowly at À0 C. After 12 h, the reaction solution was extracted with DCM three times. The organic extracts were combined, dried with anhydrous Na 2 SO 4 , and evaporated. The products 6a-e were finally purified by recrystallisation with methanol.   176.1, 163.9, 163.4, 157.4, 150.5, 124.6, 124.0, 120.4,  113.2, 105.1, 55.9.

7-Methoxy-N-(2-(3-((4-methoxybenzyl)oxy)-2-methyl-4-oxopyridin-1(4H)-yl) ethyl)-4-oxo-4H-chromene-3-carboxamide (7d)
Yield: 32%, yellow oil, 1  General procedure for the preparation of compounds 8a-e, 15a-g, and 17a-l The chromone derivatives 7a-e, 14a-g, and 16a-l (0.3 mmol) were dissolved in anhydrous DCM (10 ml) and protected by nitrogen. After the solution was cooled to À48 C, BCl 3 (1 M in DCM, 1.5 eq) dissolved in anhydrous DCM (10 ml) was added dropwise slowly. After the addition was completed, the mixture was stirred for 12 h at room temperature. The excess boron trichloride was quenched with methanol (10 ml) and left to stir for another 1 h. After the removal of solvents in a high vacuum, the residues were purified by recrystallisation from methanol/ether to afford the final compounds 8a-e, 15a-g, and 17a-l as solids.  13    General procedure for the preparation of compounds 10 and 11 The synthesis procedure of compound 10 was the same as that of compounds 5a-l. A mixture of compound 10 (10 mmol), and triethylamine (20 mmol) in anhydrous DCM (80 ml) was cooled to 0 C for a few minutes. Then acetyl chloride (10 mmol) dissolved in anhydrous DCM was added dropwise. After the addition was finished, the reaction mixture was stirred for 3 h at room temperature. The reaction solution was washed by water three times, evaporated under vacuum, and purified by column chromatography hexane/ethyl acetate. The target product 11 was obtained by removing the solvent in vacuo. General procedure for the preparation of compounds 12 and 13 The synthesis procedure of compound 12 was the same as that of compounds 6a-e. A solution of intermediate 12 (1.0 mmol) in anhydrous CH 2 Cl 2 (10 ml) was treated with DMF (2 drops). After stirring for a few minutes at room temperature, oxalyl chloride (2.0 mmol) was added dropwise to the above system. For the generation of corresponding acyl chloride, the mixture was allowed to stir for further 3 h. Subsequently, the reaction solvent was evaporated, dissolved by anhydrous DCM renewedly, and added dropwise to a solution of the intermediate 3 (2 mmol) and triethylamine (4 mmol) in anhydrous CH 2 Cl 2 at 0 C. After stirring for another 3 h at room temperature, the crude product was afforded by washed with water three times, dried, and concentrated in vacuo. The target compound was purified by column chromatography DCM/MeOH; the solvents were concentrated to give 13. Then the mixture was evaporated and neutralised by adding 1 N HCl dropwise till no more solids precipitated. Filtered, washed with water to yield a faint yellow solid. Subsequently, appropriate benzyl bromide (1.2 eq) and K 2 CO 3 (1.3 eq) were added to a solution of above intermediate (1 mmol) in DMF (4 ml). After stirring at 80 C for another 12 h, the mixture was poured into water and extracted with dichloromethane three times, and washed with water. The collected organic layers were removed under vacuum to obtain yellow oil, which was purified by chromatography (DCM: MeOH ¼ 30:1) to obtain the final compounds.
1.0, 2.0, 4.0, 6.0, 8.0, 10, 12, and 24 h post-dosing. The collected blood samples were immediately centrifuged at 3500 rpm for 10 min at room temperature to separate plasma, and stored in a À80 C refrigerator before use. For brain tissue distribution investigation, rats were sacrificed at C max time point after i.g. administration (15 mg/kg) of 17d. The tissue was weighed and homogenised under ice bath conditions and was collected for further processing.
Biological samples were performed with ACQUITY UPLC V R H Class system, which was coupled to an Xevo TQ-S micro triple quadrupole mass spectrometer (Waters, Milford, MA, USA) with an ESI source and MassLynx TM Workstation software version 4.2 (Waters, Milford, MA, USA). We used a BEH Shield RP C18 column (100 Â 2.1 mm, 1.7 mm, Waters, Milford, MA, USA) for chromatographic separation, and the column temperature is kept at 30 C. The mobile phase was composed of 30%, 0.2% formic acid aqueous solution (A) and 70%, 0.3% formic acid methanol solution (B) and is delivered at a flow rate of 0.2 ml/min. The temperature of the autosampler is set to 30 C. The sample injection volume is 5 ml. Pharmacokinetic parameters of the analytes were calculated using the pharmacokinetic software DAS version 2.0 (Bontz Inc., Beijing, China). All data were expressed as mean ± SD.

Disclosure statement
No potential conflict of interest was reported by the author(s).